Oropharynx RNA Extraction in Microcentrifuge Tubes

Buffer Preparation

• Lysis Buffer: Ready to use.
• Binding Buffer: Add 24.5 mL isopropyl alcohol to the RNA Binding Buffer bottle.
• Wash Buffer 1: Add 22.5 mL of 100% ethanol to the Wash Buffer 1 bottle.
• Wash Buffer 2 (75% ethanol): 3 parts of 100% ethanol to 1-part of molecular grade water, prepare fresh each day, producing 2.3 mL per sample with 10% excess.
• DNase Reaction Buffer: Ready to use.
• RNA Elution Buffer: Ready to use.

Enzyme Reconstitution

Proteinase K:

• Bring proteinase K vial to room temperature.
• Remove stopper and add 1.1 mL of molecular grade water to the proteinase K vial.
• Replace stopper and incubate at room temperature for 15 minutes, mixing well by inversion.
• Aliquot into required volumes and store at -20°C. Avoid multiple freeze thaw cycles

Homogenization

1. Add 500 μL of Lysis Buffer and 20 μL proteinase K into each lysis kit tube.
2. Weigh up to 50 mg of tissue samples and transfer to the buffer-filled tubes.
3. Set the Bullet Blender at speed 12, time 3 minutes and homogenize the samples. If using other homogenizer models, refer to the manufacturer's instructions for settings.
4. Remove the tubes and visually inspect the samples to confirm complete homogenization (Figure 1).

Extraction

Nucleaic Acid Binding:

1. Prepare fresh microtubes in the tube rack with 200 μL of PBS.
2. Transfer 320 μL of the homogenate to the new tubes containing PBS.
3. Centrifuge tubes at 13,000 x g for 5 minutes.
4. Transfer 450 μL of supernatant into new microfuge tubes, being careful not to disturb the pellets or transfer any debris.
5. Add 450 μL (or volume equal to the transferred supernatant from step 4) of Binding Buffer to the supernatant in each tube. Slide the tube rack onto the mixing base and invert to mix the buffer with the homogenate.
6. Vortex the MAGneat beads thoroughly making sure that there are no clumps in the tube.
7. Slide the tube rack off the mixing base and add 30 μL of the beads to each sample tube. Mix tubes by inversion to resuspend the magnetic beads. Allow to stand for 5 minutes, mixing again halfway through.
8. Slide the tube rack onto the magnetic stand and allow to sit for 1-2 minutes, until the magnetic beads separate from the homogenate (Figure 2).
9. Using a pipette, discard the supernatant without disturbing the beads (Figure 3). Remove the tube rack from the magnetic stand.

Washing I:

1. Add 700 µL of Wash Buffer 1 to the tubes and then place the tube rack in the mixing base. Mix thoroughly by inversion, ensuring all beads have been detached from the tube walls and evenly suspended (Figure 4).
2. Remove the tube rack from the mixing base and place it on the magnetic stand. Allow the magnetic beads to separate.
3. Using a pipette, discard the supernatant.
4. Remove the tube rack from the magnetic stand. Add 700 µL of Wash Buffer 2, mix thoroughly by inversion to completely resuspend the beads.
5. Transfer the tube rack to the magnetic stand and allow the magnetic beads to separate.
6. Discard the supernatant.
7. Repeat steps 4-6 for a total of 2 washes with Wash Buffer 2.
8. Centrifuge samples at 2,000 x g for 3 minutes to collect the remaining Wash Buffer. Place the tubes on the tube rack.
9. Use a 20 µL pipette to remove the remaining Wash Buffer from the tubes (Figure 5).
10. Leave tube caps open to air dry the magnetic beads at room temperature for 5 minutes.

Elution and DNase I Treatment:

1. Add 100 μL of RNA Elution Buffer to each tube. Mix by tapping to ensure that the magnetic beads are completely resuspended.
2. Prepare the DNase I mastermix. Each sample requires 100 μL of DNase I reaction buffer and 4 μL of DNase I. Mix by pipetting up and down. Do NOT vortex.
3. Add 104 μL of DNase I mastermix to each sample and mix by pipetting up and down to fully resuspend the magnetic beads.
4. Incubate samples at room temperature for 10 minutes.

Washing II:

1. Add 200 µL of RNA Binding Buffer to each tube. Mix well by inversion, ensuring all beads are resuspended.
2. Add 400 µL of Wash Buffer 2, mix well by inversion.
3. Place the tube rack onto the magnetic stand and allow the magnetic beads to separate.
4. Discard the supernatant then remove the tube rack from the magnetic stand.
5. Add 400 µL of Wash Buffer 2 and mix well by inversion. Ensure beads are detached from the tube walls.
6. Place the tube rack onto the magnetic stand and allow the magnetic beads to separate.
7. Discard the supernatant then remove the tube rack from the magnetic stand.
8. Centrifuge samples at 2,000 x g for 3 minutes to collect the remaining Wash Buffer. Place the tubes on the tube rack.
9. Use a 20 µL pipette to remove the Wash Buffer from the tubes. Do not disturb the magnetic beads.
10. Leave caps open to air dry the magnetic beads at room temperature for 5 minutes.

Elution II:

1. Add 100 µL of RNA Elution Buffer to each tube. Mix by tapping to ensure that the magnetic beads are completely resuspended.
2. Place the tube rack onto the magnetic stand and allow the magnetic beads to separate.
3. Transfer the clear supernatant containing the RNA into new, labeled microfuge tubes for downstream processing. Be careful not to transfer any magnetic beads.
4. Analyze RNA quality (OD₂₆₀/OD₂₈₀) and yield using a NanoDrop™ or other spectrophotometer and gel.
5. Isolated RNA can be stored at -80°C.